An X-ray microscope can observe a specimen in an aqueous solution at a higher resolution compared with an optical microscope. Particularly, soft X-ray having a wavelength in a range of 2.3 to 4.4 nm (corresponding to 284 to 540 eV), which is generally called as the “water window”, has a characteristic that its absorption coefficient significantly differs among materials constituting a living body, and that while it passes through water, it hardly passes through protein etc. due to large absorption by carbon and nitrogen.
Using such soft X-ray in the “water window” region will allow an object containing water (a living body specimen and a specimen in a solution) to be observed as it is, and in addition to that, will allow observation at a higher resolution than that of an optical microscope since its wavelength is shorter than visible light. From that reason, development of a soft X-ray microscope utilizing X-ray in the wavelength region of “water window” has been promoted (for example, Non Patent Literature 1: MAJIMA et. al, “Observation of living cells with X-ray Microscopy,” Medical Imaging Technology, Vol. 17, No. 3, p. 211-216, (1999)).
Besides the soft X-ray in the wavelength range of “water window”, a soft X-ray in the “carbon window” wavelength region (5.0 to 4.5 nm), which exhibits lower absorption by carbon, and a soft X-ray in a further shorter wavelength region (0.6 to 2.3 nm) are effective in the observation of biological specimens.
X-ray microscopy is primarily classified into a method of narrowing down an X-ray beam by using a condensing system such as a zone plate etc. and applying it to a specimen (a light condensing system), and a method of applying an X-ray beam from a point source to a specimen (a point light source system).
X-ray microscopes of light condensing system are classified into a radiation transmission type and a scanning transmission type (Non Patent Literature 2: Chris Jacobsen, “Soft x-ray microscopy,” Trend in Cell Biology, Vol. 9, p. 44-47 (1999)), and the resolution in this method depends on the processing accuracy of the zone plate, and a theoretical limit thereof is predicted to be 10 to 15 nm (Non Patent Literature 3: W. Chao et al., “Soft X-ray microscopy at a spatial resolution better than 15 nm,” Nature, Vol. 453, p. 1210-1213 (2005)).
On the other hand, while the method of utilizing a point light source is classified into a method of generating X-ray with a laser and a method of generating X-ray with an electron beam, a method of observing a specimen with X-ray, which is generated by making an electron beam incident on a target, as a probe is being developed.
This method adopts a technique in which an electron beam is made directly incident on a specimen supporting film to generate X-ray, and the X-ray is made to irradiate a specimen adhered to the specimen supporting film opposite the electron beam incident direction (Patent Literature 1: Japanese Patent Laid-Open No. 8-43600, Patent Literature 2: Japanese Patent Application No. 2009-18290 specification, and Patent Literature 3: Japanese Patent Laid-Open No. 2-138856).
According to such a technique, since a charged particle beam which is narrowed to be extremely thin is made incident on a specimen supporting film thereby allowing the diffusion range of the charged particles to be suppressed, it is possible to achieve a high resolution. Moreover, since mounting a plurality of X-ray detectors at various angles and locations below the specimen supporting film will enable the acquisition of inclined images depending on the mounting angle thereof, inclined images of the same number as that of the detectors are obtained by one charged beam scanning, making it possible to recreate a three-dimensional structure of the specimen to be observed (Patent Literature 2: Japanese Patent Application No. 2009-18290 Specification).
As the specimen supporting member (supporting film) to be used for such soft X-ray microscope observation, a silicon nitride film has been widely used. Since the silicon nitride film is excellent in pressure resistance, even when it is provided at a window portion of the specimen containing cell of which interior is under atmospheric pressure, it will cause no hindrance for use in a microscope apparatus which is to be put under vacuum. For example, in the invention disclosed in Japanese Patent Laid-Open No. 6-180400 (Patent Literature No. 4), a specimen cell in which two sheets of silicon nitride film are fixed in parallel with a predetermined interval therebetween, and an observation specimen is contained in the spacing with aqueous solution to be sealed is introduced into a soft X-ray microscope apparatus of which interior is under vacuum.
Meanwhile, when a specimen in an aqueous solution is observed at a high resolution, it is necessary to fix the specimen to the supporting member to suppress the deflections of an observed image caused by Brownian motion, and it is an essential factor that such a supporting film for fixing the specimen to the specimen supporting member is as thin as possible and highly durable, and further has a high transparency to X-ray.
Conventionally, organic matters such as concanavalin A and polyimidines have been used as the fixing agent for a biological specimen. However, since such organic matters contain a large amount of carbon and nitrogen, soft X-ray in the above described water window region is apt to be absorbed, thereby significantly reducing the contrast of a resulting observation image. Moreover, since the molecular size is as large as several tens of nm, a problem also exists in that the structure of such specimen fixing agent itself is observed at the time of high resolution observation. Further, they also have drawbacks that they are susceptible to external environments such as heat and ultra violet ray or humidity, and are lack of durability.